Vibrio parahaemolyticus is an important cause of diarrheal disease due to the consumption of imporperly prepared seafood. Although the pathogenesis of disease due to this species is uncertain, a consistent finding in strains isolated from disease is the presence of a hemolysin designated thermostable direct hemolysin (TDH) or the "Kanagawa Phenomenon" (KP) associated hemolysin. Previous research on this project has resulted in the cloning and sequencing of genes encoding TDH (tdh), the development and testing of a DNA probe for tdh genes and the construction of isogenic strains which do not produce TDH. The proposed continuation of this project will answer additional questions that have arisen concerning the genetics of TDH production and investigate the role of a recently discovered shiga-like toxin in the pathogenesis of disease due to V. parahaemolyticus. The proposed research will focus on the cloning and sequencing of genes encoding the shiga-like toxin and assess the significance of this toxin in the disease process. The specific aims of this project are as follows: 1) To investigate the phenomenon of gene duplication of the gene encoding TDH (tdh). 2) To clone and sequence the single gene copy present in strains of V. parahaemolyticus which produce very low amounts of TDH and compare this gene to tdh genes found in strains which produce high amount of TDH. 3) To investigate the role of plasmids in the transfer of tdh genes. 4) To clone and characterize genes encoding a shiga-like toxin in V. parahaemolyticus. 5) To construct isogenic strains of V. parahaemolyticus which do not produce the shiga-like toxin and assess their potential pathogenicity. 6) To develop a DNA probe capable of detecting genes encoding shiga-like toxin and employ it to survey the incidence of these genes in environmental and clinical isolates of V. parahaemolyticus and in other Vibrio species pathogenic for man.